Method for purification of alpha-1-antitrypsin

ABSTRACT

A streamlined method for purifying alpha-1-antitrypsin (AAT) from an AAT-containing protein mixture, such as a Cohn fraction IV precipitate, is provided. In the method of the invention, contaminating proteins are destabilized by cleavage of disulfide bonds with a reducing reagent, such as a dithiol, which does not affect AAT. The destabilized proteins are then preferentially adsorbed on a solid protein-adsorbing material, without the addition of a salt as a precipitant. Separation of the solid adsorbent from the solution leaves a purified AAT solution that is directly suitable for chromatographic purification, without the need for extensive desalting as in prior art processes. A process incorporating this method, which provides pharmaceutical grade AAT in high yield on a commercial scale, is also described.

FIELD OF THE INVENTION

[0001] The invention relates to protein separation and purificationmethods. More specifically, the invention relates to the separation ofalpha-1-antitrypsin (AAT, also known as alpha-1 proteinase inhibitor,API, and A₁-PI) from complex protein mixtures such as blood plasmafractions, and to methods for further purification of the separated AATso as to provide a composition suitable for pharmaceutical use.

BACKGROUND OF THE INVENTION

[0002] Alpha-1-antitrypsin (AAT) is a glycopeptide inhibitor ofproteases, and is found in human serum and other fluids. Proteaseinhibition by AAT is an essential component of the regulation of tissueproteolysis, and AAT deficiency is implicated in the pathology ofseveral diseases. Individuals who inherit an alpha-1 antitrypsindeficiency, for example, have increased risk of suffering from severeearly-onset emphysema, the result of unregulated destruction of lungtissue by human leukocyte elastase. The administration of exogenoushuman AAT has been shown to inhibit elastase and is associated withimproved survival and reduction in the rate of decline of lung functionin AAT-deficient patients (Crystal et al., Am. J. Respir. Crit. CareMed. 158:49-59 (1998); see R. Mahadeva and D. Lomas, Thorax 53:501-505(1998) for a review.)

[0003] Because of its therapeutic utility, commercial AAT production hasbeen the subject of considerable research. Much progress has been madein the production of recombinant AAT in E. coli (R. Bischoff et al.,Biochemistry 30:3464-3472 (1991)), yeast (K. Kwon et al., J.Biotechnology 42:191-195 (1995); Bollen et al., U.S. Pat. No.4,629,567), and plants (J. Huang et al., Biotechnol. Prog. 17:126-33(2001)), and by secretion in the milk of transgenic mammals (G. Wrightet al., Biotechnology, 9:830-834 (1991); A. L. Archibald, Proc. Natl.Acad. Sci. USA, 87:5178-5182 (1990)). However, isolation of AAT fromhuman plasma is presently the most efficient practical method ofobtaining AAT in quantity, and human plasma is the only FDA-approvedsource.

[0004] A number of processes for isolating and purifying AAT from humanplasma fractions have been described, involving combinations ofprecipitation, adsorption, extraction, and chromatographic steps. Inorder to minimize the risk of pathogen transfer, pooled human plasmaintended for production of human AAT for therapeutic use is screened forthe hepatitis B surface antigen, and for antibodies to the humanimmunodeficiency virus. As an additional precaution against transmissionof infectious agents, the purified product is ordinarily pasteurized byheating to 60° C. for 10 hours (Mitra et al., Am. J. Med. 84(sup.6A):87-90 (1988)) and sterile filtered.

[0005] Most published processes for AAT isolation begin with one or morefractions of human plasma known as the Cohn fraction IV precipitates,e.g. Cohn fraction IV₁ or fraction IV₁₋₄, which are obtained from plasmaas a paste after a series of ethanol precipitations and pH adjustments(E. J. Cohn et al., J. Amer. Chem. Soc., 68:459-475 (1946)).

[0006] U.S. Pat. No. 3,301,842 describes a method for isolation of AATfrom Cohn fraction IV₁ wherein an acridine or quinoline derivative isadded to the paste in a buffer at pH 6, the precipitate is discarded,and the pH adjusted to 7.0. Additional acridine or quinoline is added,and the precipitate is collected. This precipitate is dissolved in a pH5.0 buffer, sodium chloride is added, and the resulting precipitatediscarded. The solution, containing the AAT, is further processed bymethanol precipitation. Alternatively, ammonium sulfate precipitationsat pH 8 and at pH 5 are conducted with plasma, with the pH 5 supernatantbeing further processed as above with quinoline or acridine additives.

[0007] Glaser et al., Preparative Biochemistry, 5:333-348 (1975),disclosed a method for isolating AAT from Cohn fraction IV₁ paste. Thepaste is stirred in a phosphate buffer at pH 8.5 in order to reactivatethe AAT, which is largely deactivated by the pH of 5.2 employed in theCohn fractionation. After dialysis and centrifugation, the supernatantis subjected to two rounds of anion exchange chromatography at pH 6.0 to7.6 and at pH 8.6, followed by further chromatographic processing at pH7.6 and at pH 8.0, to produce AAT in about a 30% overall yield.

[0008] M. H. Coan et al., in U.S. Pat. Nos. 4,379,087 and 4,439,358 (seealso M. H. Coan et al., Vox Sang., 48:333-342 (1985); M. H. Coan, Amer.J. Med., 84(sup 6A):32-36 (1988); and R. H. Hein et al., Eur. Respir.J., 3(sup 9):16s-20s (1990)), disclosed a procedure wherein Cohnfraction IV₁ paste is dissolved in a pH 6.5 to 8.5 buffer, polyethyleneglycol is added, and the pH is lowered to the range of 4.6 to 5.7 toprecipitate unwanted proteins. After centrifugation, AAT is isolatedfrom the supernatant by anion exchange chromatography. Furtherprocessing provides a 45% yield of AAT with a purity of about 60%.Methods employing polyethylene glycol as a precipitant are alsodescribed in U.S. Pat. No. 4,697,003, U.S. Pat. No. 4,656,254, andJapanese patent JP 08099999, described below; and also by Hao et al.,Proc. Intl. Workshop on Technology for Protein Separation andImprovement of Blood Plasma Fractionation, Sep. 7-9, 1977, Reston, Va.

[0009] Dubin et al., Preparative Biochemistry. 20:63-70 (1990),disclosed a two step chromatographic purification, in which AAT wasfirst eluted from Blue SEPHAROSE™ and then purified by gel filtrationchromatography.

[0010] Schultze and Heimburger, in U.S. Pat. No. 3,293,236, disclosedpurification of AAT using cation exchange chromatography with a citratebuffer, in combination with ammonium sulfate fractionation of humanplasma.

[0011] Lebing and Chen, in U.S. Pat. No. 5,610,285, disclosed apurification process which employs an initial anion exchangechromatography, followed by cation exchange chromatography at low pH andlow ionic strength, to purify human AAT from plasma and plasmafractions. The cation chromatography takes advantage of the fact thatactive AAT does not bind to the ion exchange column under theseconditions while contaminating proteins, including denatured AAT andalbumin, are retained.

[0012] Jordan et al., in U.S. Pat. No. 4,749,783, described theisolation of AAT from human plasma using affinity chromatography withmonoclonal antibodies. See also Podiarene et al., Vopr. Med. Khim.35:96-99 (1989).

[0013] Shearer et al., in European patent application EP 0 224 811 andin the corresponding U.S. Pat. No. 4,656,254, disclosed an improvedmethod for extracting AAT from Cohn fraction IV paste, in which theimprovement consisted of treating the paste with a larger volume ofbuffer, at a higher pH, than had been customary in the prior art. Thecombination of higher volume and higher pH increased the amount of AATextracted from the paste. Unwanted proteins were precipitated byaddition of polyethylene glycol, followed by centrifugation. Analternative procedure is disclosed, which is essentially the procedureof Coan et al., wherein after addition of polyethylene glycol, the pH isadjusted to the range of 4.6 to 5.7, and the acidified mixture held forfrom one to sixty minutes to further precipitate unwanted proteins. TheAAT is precipitated by addition of additional polyethylene glycol, andfurther purified by anion exchange chromatography.

[0014] Arrighi et al., in European application EP 0717049, disclosed aprocess wherein fraction IV₁ paste is stirred in a pH 8.2 buffer at 40°C. for one hour, followed by precipitation of unwanted proteins withammonium sulfate. The AAT is isolated from the supernatant byhydrophobic interaction chromatography at pH 7.

[0015] Kress et al., in Preparative Biochemistry 3:541-552 (1973),dialyzed the precipitate from an 80% ammonium sulfate treatment of humanplasma, then chromatographed it on DEAE-cellulose. The product wasdialyzed again and gel filtered on SPEHADEX™ G-100. AAT-containingfractions were then chromatographed on DE-52 cellulose to give AAT.

[0016] Japanese patent 59-128335 discloses the precipitation of unwantedproteins from a plasma fraction by addition of polyethylene glycol at apH between 5 and 7, followed by anion exchange chromatography.

[0017] Bollen et al., in U.S. Pat. No. 4,629,567, disclose the isolationof AAT from a culture of yeast carrying recombinant plasmids expressingAAT. The process begins with polyethylene glycol precipitation at pH 6.5to remove contaminating proteins, followed by anion exchangechromatography at pH 6.5 and subsequent chromatographic steps.

[0018] Dove and Mitra, in U.S. Pat. No. 4,684,723, disclose a variant ofthe method of Coan et al. (U.S. Pat. No. 4,379,087 and U.S. Pat. No.4,439,358) in which AAT is purified by a process comprising the steps of(a) holding a solution containing AAT at a pH of 6.5 to 8.5 for up to 24hours, (b) adding polyethylene glycol and an inorganic salt, so as toobtain a two-phase mixture, and (c) isolating the aqueous salt phase,which contains purified AAT.

[0019] Taniguchi et al., in PCT application WO 95/35306, disclose asimilar process, involving precipitation with polyethylene glycol in thepresence of zinc chloride, followed by anion-exchange chromatography andchromatography on a metal chelate resin.

[0020] Van Wietnendaele et al., in U.S. Pat. No. 4,857,317, alsodisclose a process for isolating AAT from the crude extract of anengineered yeast culture, which comprises addition of polyethyleneglycol at pH 6.1, centrifugation to remove precipitated proteins,addition of calcium chloride, storage for 24 hours at pH 7.0, andcentrifugation to further remove contaminants. AAT is then isolated fromthe supernatant by subsequent chromatographic steps.

[0021] Coan, in U.S. Pat. No. 4,697,003, discloses a method forisolating AAT from various Cohn plasma fractions which comprises theremoval of ethanol and salts from an AAT-containing fraction, followedby anion-exchange chromatography on DEAE cellulose or a similar materialunder conditions such that the AAT is retained on the column whileundesired proteins are eluted. Coan also describes “pasteurization” atabout 60° C. or more for about 10 hours, which is stated to besufficient to render hepatitis viruses non-infective.

[0022] Coan discloses addition of carbohydrate as a stabilization agent,either alone or with sodium citrate, in order to stabilize the AAT atthe pasteurization temperature. Suitable carbohydrates are said to bemono-, di-, and trisaccharides, and sugar alcohols such as sorbitol andmannitol. AAT is prone to both polymerization and to the adoption ofinactive conformations upon heating; the presence of stabilizers reducesbut does not eliminate thermal inactivation (D. Lomas et al., Eur. Resp.J. 10:672-675 (1997)). Size-exclusion HPLC analysis has shown that 10%of monomeric AAT is polymerized or aggregated when pasteurization iscarried out according to the Coan process (M. H. Coan et al., Vox Sang.,48:333-342 (1985)).

[0023] T. Bumouf et al., Vox Sang., 52:291-297 (1987), disclosedsubstantially the same procedure for isolating AAT fromKistler-Nitschmann supernatant A. DEAE chromatography of Cohn FractionsII+III and size exclusion chromatography produced an AAT which was80-90% pure (by SDS-PAGE) with a 36-fold increase in purity. Recoverywas 65-70%.

[0024] Thierry, in European patent application EP 0282363, alsodiscloses a method of obtaining AAT from a Kistler-Nitschmann plasmafraction. Briefly, plasma is precipitated with 10% ethanol at pH 7.4,and the supernatant precipitated again with 19% ethanol at pH 5.85. Thesupernatant from the second precipitation is applied to a DEAEanion-exchange column, and eluted at pH 5.2 to provide AAT of about 90%purity.

[0025] Strancar et al., in PCT patent application WO 95/24428, disclosea very similar method, employing a particular class of functionalizedanion-exchange media. Desalted Cohn fraction IV₁ is applied to thecolumn, and contaminating proteins are eluted with low salt buffer at apH “close to the pKa of acetic acid.” (The pKa of acetic acid is 4.74.)AAT is then eluted with 50 to 300 mM NaCl at pH 7.4 to 9.2.

[0026] Japanese patent JP 08099999 discloses a method of obtaining AATfrom Cohn fraction IV or IV₁, which involves reduction of saltconcentration to below about 0.02 M, adjusting the pH to 4.5 to 5.5, andcontacting the solution with a cation exchanger to adsorb contaminatingproteins.

[0027] M. E. Svoboda and J. J. van Wyk, in Meth. Enzymology, 109:798-816(1985), disclose acid extraction of Cohn fraction IV paste withphosphoric, formic, and acetic acids.

[0028] Glaser et al., in Anal. Biochem., 124:364-371 (1982) and also inEuropean Patent Application EP 0 067 293, disclose several variations ona method for isolating AAT from Cohn fraction IV₁ precipitate whichcomprises the steps of (a) dissolving the paste in a pH 8.5 buffer, (b)filtering, (c) adding a dithiol such as DTT, and (d) precipitation ofdenatured proteins with ammonium sulfate. Glaser states that thedestabilized (DTT-reduced) proteins may be precipitated by “suitabletechniques such as salting, heating, change in pH, addition of solventsand the like.”

[0029] Glaser et al. describe one variation in which treatment with DTTis carried out in the presence of 2.5% AEROSIL™ fumed silica, prior toprecipitation with 50% saturated ammonium sulfate. Recovery of AAT wasas good as it was in the absence of the silica, and the purificationfactor was improved by about 70%. In both references, the authorsrelegate the silica to a secondary role, that of an additive thatimproves the results of the ammonium sulfate precipitation. Theeffectiveness of silica alone, without ammonium sulfate precipitation,is not recognized or described. If the concentration of the proteinsolution appreciably exceeds about 50 mg protein/ml, AAT is reportedlylost by occlusion in the precipitate.

[0030] Ralston and Drohan, in U.S. Pat. No. 6,093,804, disclose a methodinvolving the removal of lipoproteins from an initial protein suspensionvia a “lipid removal agent,” followed by removal of “inactive AAT” viaelution from an anion-exchange medium with a citrate buffer. The lipidremoval agent is stated to be MICRO CEL™ E, a synthetic hydrous calciumsilicate. In the presence of a non-citrate buffer, the anion-exchangemedium binds active AAT while allowing “inactive AAT” to pass through. Acitrate buffer is specified for subsequent elution of the AAT from theanion exchange medium, and also for later elution from a cation-exchangemedium. Ralston and Drohan do not describe the use of adisulfide-reducing agent. The process is stated to provide AAT with aproduct purity of >90%; and manufacturing scale yields of >70%.

[0031] W. Stephan, in Vox Sanguinis 20:442-457 (1971), describes the useof fumed silica to adsorb lipoproteins from human blood serum solutions.The effect of silica adsorption on the concentrations of several plasmaproteins, including AAT, was evaluated, and there was no significantloss of AAT.

[0032] Mattes et al., in Vox Sanguinis 81:29-36 (2001), and in PCTapplication WO 98/56821 and published US patent application2002/0082214, disclose a method for isolating AAT from Cohn fraction IVwhich involves ethanol precipitation, anion exchange chromatography, andadsorption chromatography on hydroxyapatite. The latter step is reportedto remove inactive AAT, providing a product with very high specificactivity.

[0033] While AAT is an effective treatment for emphysema due toalpha-1-antitrypsin deficiency, treatment is very costly (currentlyabout $25,000 per year), due to the limited supply and a complexmanufacturing process. There remains a need for more efficient andcost-effective methods for isolating human AAT from plasma and othercomplex protein mixtures containing AAT. In particular, ammonium sulfateprecipitation followed by dialysis is a time-consuming process, thatgenerates substantial amounts of waste water, and there is a need forscalable processes that do not require extensive dialysis whileproviding high yields of high activity, high purity AAT. Thermalpasteurization of AAT effectively reduces viral contamination, but itleads to the formation of inactive AAT aggregates and polymers. Thus,there is also a need for highly pure AAT with reduced viralcontamination but without significant amounts of inactive AAT aggregatesand polymers. The present invention addresses these needs.

BRIEF DESCRIPTION OF THE INVENTION

[0034] The invention provides a method for purifying AAT from crudeAAT-containing protein precipitates, which consists essentially of thefollowing steps: (a) suspending the AAT-containing protein mixture in abuffer under conditions that permit the AAT to be dissolved; (b)contacting the resulting suspension with a disulfide-reducing agent toproduce a reduced suspension; (c) contacting the reduced suspension withan insoluble protein-adsorbing material; and (d) removing insolublematerials from the suspension. This process provides an enriched AATpreparation, directly suitable for chromatographic processing, withreduced costs and in less time than prior art processes. Additionalpurification steps may be performed at the discretion of thepractitioner, as described further below.

[0035] More specifically, the process comprises the steps of: (a)suspending a crude AAT-containing protein precipitate in a buffer underconditions that permit the AAT to be dissolved; (b) contacting theresulting suspension with a disulfide-reducing agent, under conditionsthat permit reduction of intra-protein disulfide bonds by the reducingagent, to produce a reduced suspension; (c) contacting the reducedsuspension with an insoluble protein-adsorbing material, without theaddition of a substantial amount of additional salts and (d) removinginsoluble materials from the suspension, so as to obtain a clarifiedprotein solution.

[0036] By “substantial amount of additional salts” is meant an amount ofsoluble salt or salts that will cause otherwise-soluble proteins tobegin precipitating from the solution in significant amounts. Thosesalts ordinarily used to cause any degree of protein precipitation, inthe amounts ordinarily used for such purposes, are specificallyincluded.

[0037] The method of the invention eliminates the salting-out step whichwas taught by Glaser in EP 0 067 293, which in turn avoids the time andcost associated with the need to desalt the filtrate by extensivedialysis. Furthermore, the ammonium sulfate precipitation employed byGlaser limited the concentration of the protein solutions that could beprocessed. If the protein concentration appreciably exceeds about 50mg/ml in Glaser's method, AAT is reportedly lost by occlusion in theAerosil/protein precipitate. In the absence of ammonium sulfate, higherconcentrations of protein should be usable without precipitation andocclusion of AAT, with associated savings in reagents and processingtime, and greater throughput per batch. The process of the presentinvention involves two steps where protein concentration exceeds 100mg/ml in the absence of ammonium sulfate, and no precipitation of AAThas been seen.

[0038] The combination of a disufide-reducing agent and an insolubleprotein-adsorbing material according to the invention is particularlyeffective at removing albumin and transferrin, which are the majorprotein impurities in serum-derived crude AAT preparations such as Cohnfraction IV precipitates. After removal of the protein-adsorbingmaterial by filtration, both albumin and transferrin levels are belowthe detection limits of nephelometry when conducted as described herein.Further processing as described herein provides AAT with an averagepurity of 98% by SDS-PAGE (reduced), and high specific activity,averaging 1.06 mg functional AAT/mg. Compositions with purity greaterthan 99% by SDS-PAGE, and having specific activities up to 1.12 mgfunctional AAT/mg protein, can be obtained by the methods disclosedherein.

[0039] The crude AAT-containing protein precipitate may be derived fromvarious sources, including but not limited to human serum, serum from atransgenic mammal that expresses human AAT, or milk from a transgenicmammal that secretes human AAT in its milk. The source is preferablyserum. If the source is serum, the precipitate is preferably a Cohnfraction IV precipitate, more preferably Cohn fraction IV₁, and mostpreferably Cohn fraction IV₁₋₄. There are variations, known to those ofskill in the art, in the method for preparing Cohn fractions, and any ofthem may be employed in the present invention.

[0040] The suspension buffer may be any aqueous buffer in which AAT issoluble, and is used in a volume sufficient to dissolve most or all ofthe AAT present in the precipitate. The preferred volume for suspensionof Cohn fraction IV₁₋₄ is between 6 and 10 liters per kg of precipitatepaste. Examples of buffers include, but are not limited to, citrate,phosphate, and Tris buffers. The preferred buffer is Tris, preferably100 mM Tris with 20 mM NaCl. The preferred pH is between 8.80 and 8.95.

[0041] The disulfide-reducing agent may be any dithiol commonly used toreduce disulfide bonds in proteins, including but not limited todithiothreitol (DTT), dithioerythritol (DTE), 1,2-ethanedithiol,1,2-propanedithiol, 1,3-propanedithiol, and the like; or a phosphinesuch as tributylphosphine or trimethylphosphine. The disulfide-reducingagent is preferably a dithiol, and most preferably dithiothreitol.

[0042] The insoluble protein-adsorbing material may be any of variousknown adsorbents for hydrophobic proteins, such as fumed silica; silicahydrogels, xerogels, and aerogels; calcium, aluminum and magnesiumsilicates; certain clays or minerals; and mixtures thereof. Suchmaterials are commonly used for the clarification of food oils andbeverages, and are well-known to those of skill in the art. Preferablythe protein-adsorbing material is a silica adsorbent, more preferably afumed silica such as that sold under the trade name AEROSIL™.

[0043] The invention also provides a novel combination of purificationand virus reduction and inactivation steps, which produces a high-safetyand high-purity AAT suitable for pharmaceutical use. Specifically, whilethe use of dithiothreitol and fumed silica in AAT purification processeshas been described previously, the combination of the two in the absenceof high temperatures or a precipitating agent such as ammonium sulfatehas not been described previously. Surprisingly, it has been found thatthe omission of a precipitating agent from a dithiothreitol-AEROSIL™treatment step provides a highly effective purification stage.Furthermore, while the uses of dithiothreitol, AEROSIL™, anion exchangechromatography, hydrophobic interaction chromatography, pasteurization,and nanofiltration have each been previously described in theliterature, these particular steps are now combined for the first timein a purification process suitable for industrial manufacture ofpharmaceutical grade AAT.

[0044] The present invention provides a preparation of AAT characterizedby the following properties:

[0045] (a) the alpha-1-antitrypsin contains less than 6%, preferablyless than 2%, and most preferably less than 1% contaminating proteins bySDS-PAGE, and contains

[0046] (b) less than 0.1% Albumin;

[0047] (c) less than 0.8%, and preferably less than or equal to 0.2%α₁-acid glycoprotein;

[0048] (d) less than 0.1% α₂-macroglobulin;

[0049] (e) less than 0.1% apolipoprotein A1;

[0050] (f) less than 0.5%, and preferably less than or equal to 0.1%antithrombin III;

[0051] (g) less than 0.1% ceruloplasmin;

[0052] (h) less than 0.5%, and preferably less than 0.1% haptoglobin;

[0053] (i) less than 0.2%, and preferably less than 0.1% IgA;

[0054] (j) less than 0.1% IgG;

[0055] (k) less than 0.1%. transferrin;

[0056] (l) the specific activity of the alpha-1-antitrypsin is at least0.99 mg functional AAT/mg, when using as an extinction coefficientE_(1 cm,280 nm) ^(1%)=5.3;

[0057] (m) less than 8%, and preferably less than 5%, of the product isof a higher molecular weight than monomeric AAT;

[0058] (n) the apparent ratio of active to antigenic AAT is greater than1.08, preferably greater than 1.16, and most preferably greater than1.23, when measured by endpoint nephelometry;

[0059] (o) enveloped viruses are reduced by at least 11 log₁₀ units, andnon-enveloped viruses by at least 6 log₁₀ units, when measured inspiking studies using human and model viruses representing a wide rangeof physico-chemical properties; and

[0060] (p) the product is stable for at least 2 years when storedlyophilized at up to 25° C.

[0061] The apparent ratio of active to antigenic AAT in the product ofthe present invention is greater than unity because the purity and/oractivity of the product of the present invention is greater than that ofthe reference standard, which is a prior art composition. Antigeniclevels, as determined by endpoint nephelometry, are measured against thecurrent protein standard (product No. OQIM15, supplied by Dade-Behring,Deerfield, Ill.), which is calibrated directly against theinternationally-recognized Certified Reference Material 470 (ReferencePreparation for Proteins in Human Serum; see J. T. Whicher et al., Clin.Chem. 40:934-938 (1994)), using reagents and AAT antibody (Dade-Behringproduct No. OSAz15), as supplied for the Dade-Behring Nephelometer 100.

[0062] All publications and patent applications specifically referencedherein are incorporated by reference in their entirety. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. The term “AAT” refers to human AATgenerally, whether heterogeneous or homogeneous, and whether isolatedfrom human serum or from a recombinant organism. The term is intended toembrace pharmacologically effective naturally-occurring variants (seefor example, Brantly et al., Am. J. Med. 84(sup.6A):13-31 (1988)), aswell as pharmacologically effective non-natural forms of human AAT,including but not limited to those having non-human glycosylationpatterns, N-terminal methionine, or altered amino acids. Those of skillin the art will appreciate that methods and materials similar orequivalent to those described herein can be used in the practice of thepresent invention, and such equivalents are anticipated to be within thescope of the invention. The preferred embodiments described below areprovided by way of example only, and the scope of the invention is notlimited to the particular embodiments described.

BRIEF DESCRIPTION OF THE FIGURES

[0063]FIG. 1 is a flow chart showing an overall AAT purification processthat incorporates the present invention.

[0064]FIG. 2 is an SDS-PAGE gel showing the proteins present in theproducts produced by the process of the invention at various stages.Lane 1, molecular weight markers; Lane 2, Plasma (Cryo-Poor); Lane 3,Fraction IV_(1,4) Extract; Lane 4, DTT/Aerosil-Treated Extract Filtrate;Lane 5, IEC Eluate; Lane 6, HIC Effluent; Lane 7, final container.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The particular embodiment of the invention exemplified belowemploys a particular Cohn fraction IV paste as a starting material, butthe use of similar plasma fractions is contemplated to be within thescope of the present invention. Alternative starting materials includebut are not limited to other AAT-containing Cohn fractions (see U.S.Pat. No. 4,697,003), a precipitate from Kistler-Nitschmann supernatantsA or A+I (P. Kistler, H. S. Nitschmann, Vox Sang., 7:414-424 (1962)),and ammonium sulfate precipitates from plasma as described by Schultzeet al. in U.S. Pat. No. 3,301,842. The use of protein precipitatesderived from cultures of AAT-producing recombinant cells or organisms,or precipitates derived from the milk or serum of transgenic mammals, isalso contemplated to be within the scope of the present invention.

[0066] There are many methods known in the art for selectivelyprecipitating proteins from solution, such as by the addition of salts,alcohols, and polyethylene glycol, often in combination with cooling andvarious pH adjustments. It is anticipated that the present inventionwill be applicable to most AAT-containing protein precipitatescontaining recoverable AAT activity, regardless of how they areinitially prepared. The term “crude AAT-containing protein precipitate”is used herein to refer to any AAT-containing protein precipitateprepared by one or more of these known methods, whether from serum,milk, cell culture, or other original source.

[0067] In a preferred embodiment, described below, the crudeAAT-containing protein precipitate is suspended in a Tris buffer, andtreated with dithiothreitol (DTT, a preferred disulfide-reducing agent)and fumed silica (a preferred protein-adsorbing material) in order toremove contaminating proteins and lipids. Where the precipitate is Cohnfraction IV, the two major protein contaminants thus removed are albuminand transferrin. DTT and other dithiols, as well as phosphines, areknown in the art to reduce intrachain and inter-chain disulfide bonds.Cleavage of structurally important disulfide bonds causes partialunfolding and destabilization of those contaminating proteins that havedisulfide bonds. AAT itself is not destabilized by DTT treatment becauseit has no intrachain disulfide bonds.

[0068] Fumed silica is known to bind preferentially to hydrophobicproteins. It is theorized that in the method of the invention, thedestabilized contaminating proteins bind to a protein-adsorbing materialsuch as fumed silica because the partial unfolding caused by disulfidebond cleavage exposes the proteins' inner core of hydrophobic residues.The scope of the invention is not limited, however, to any particulartheory of operation.

[0069] In a preferred embodiment, described below, the protein-adsorbingmaterial, together with the adsorbed contaminating proteins, lipids, andother insoluble material, is removed from the suspension by filtrationso as to obtain a clarified AAT-containing protein solution. Filtrationis preferably carried out with the assistance of a filtering aid such asCelite™ diatomaceous earth, and preferably the suspension isrecirculated through the filter until a clarity of <10 nephelometerturbidity units (NTU)/ml is achieved. The filtrate is further processedby chromatographic techniques to afford highly pure and highly activeAAT. Other methods of separation known in the art, for examplecentrifugation, could also be employed in place of filtration. Thepractitioner will select the method appropriate to the scale ofoperations and the nature of the protein-adsorbing material.

[0070] After removal of insoluble materials, the AAT-containing solutionmay be further processed by any of the methods known in the art forprotein purification, particularly the methods already known to besuitable for purification of AAT. In a preferred embodiment describedbelow, the filtrate is first subjected to ion exchange chromatography(“IEC”) with salt gradient elution. The chromatography column containsan anion exchange resin which consists of a porous resin support matrixto which positively charged groups are covalently attached. Thesepositively charged groups reversibly bind anions, including proteinswith anionic groups such as AAT.

[0071] AAT, and other proteins which have a net negative charge at thepH of the eluting buffer, bind to the IEC column. Contaminating proteinshaving little or no negative charge pass through the anion exchangeresin column without binding and exit with the column effluent. Thosecontaminating proteins that do bind to the column are then separatedfrom the AAT by gradient elution. The salt concentration is graduallyincreased as the column is eluted in order to release sequentially thevarious proteins that are bound to the resin.

[0072] In a preferred embodiment, described below, the AAT-containingeluate from the IEC column is subjected to hydrophobic interactionchromatography (“HIC”). This type of chromatography employs a supportmatrix to which moieties are covalently attached. In an aqueousenvironment, these hydrophobic moieties bind reversibly to hydrophobicmolecules, such as the contaminating proteins remaining in the IECeluate. AAT is relatively non-hydrophobic, therefore the majority of theAAT flows through the column during the elution of the column withbuffer, while the more hydrophobic contaminating proteins remain boundto the column. The column effluent thus contains the purified AAT. Inpractice, AAT has been found to have a slight affinity for certain HICcolumn media, and in such cases further elution with several volumes ofwash buffer may be desirable in order to recover substantially all ofthe AAT in the originally-applied sample.

[0073] After such additional purification steps as are required to reachthe desired level of purity and activity, the AAT solution is thenconcentrated and sterilized. In a preferred embodiment, described below,the AAT is at a pharmaceutically acceptable level of purity and activityafter the hydrophobic interaction chromatography, and no additionalsteps are necessary. In a preferred embodiment, described below,concentration is accomplished by ultrafiltration followed by dialysisfiltration (diafiltration). In these techniques, solvent and dissolvedsalts and small molecules are passed through a filtering membrane,leaving behind a more concentrated protein solution. Remaining salts andsmall molecules in the protein solution are then exchanged with adifferent buffer by continuous addition of several volumes of the newbuffer to the product, while maintaining a constant product volume bycontinuously passing solution through the same membrane.

[0074] The AAT is then provided with a pharmaceutically acceptablebuffer, and lyophilized by methods known in the art, preferably bymethods known to be suitable for preparing AAT therapeutic formulations.

[0075] Proteins isolated from mammalian sources may contain pathogenicviral contaminants, and it is desirable to reduce or eliminate suchcontamination in pharmaceutical compositions. Methods of viral reductionare known to those of skill in the relevant arts. The methodscontemplated to be applicable to the present invention include, but arenot limited to, pasteurization, irradiation, solvent/detergenttreatment, disinfection, filtration, and treatment with supercriticalfluids. Solvent/detergent treatment can be carried out, for example, bycontacting a protein solution with a polyoxyethylene sorbitan ester andtributyl phosphate (see U.S. Pat. No. 4,820,805; see also WO 95/35306for application of the method to an AAT composition.) Disinfection of aprotein solution can be carried out by exposing the solution to asoluble pathogen inactivating agent, for example as disclosed in U.S.Pat. Nos. 6,106,773, 6,369,048 and 6,436,344, or by contact with aninsoluble pathogen inactivating matrix, for example as disclosed in U.S.Pat. No. 6,096,216 and references therein. Filtration may be through15-70 nm ultrafilters (e.g., VirAGard™ filters, A/G Technology Corp.;Planova™ filters, Asahi Kasei Corp.; Viresolve™ filters, MilliporeCorp.; DV and Omega™ filters, Pall Corp.) Irradiation may be withultraviolet or gamma radiation; see for example U.S. Pat. No. 6,187,572and references therein. Inactivation of viruses by treatment withsupercritical fluids is described in U.S. Pat. No. 6,465,168.Pasteurization of a protein solution may be accomplished by heatingwithin the limits dictated by the thermal stability of the protein to betreated. In the case of AAT, pasteurization is usually accomplished byheating to about 60-70° C. In a preferred embodiment, described below,viral reduction of the AAT concentrate is carried out by pasteurizationand ultrafiltration. Stabilizing additives may be added to protect theAAT from thermal degradation during the pasteurization step, asdisclosed for example in U.S. Pat. No. 4,876,241. Sucrose and potassiumacetate are preferably added as stabilizers, and the stabilized AATsolution is then pasteurized at about 60° C. to reduce viralcontamination. The amount of sucrose is preferably at least 40%, morepreferably at least 50%, and most preferably about 60% by weight. Use ofless than 40% sucrose has been found to result in undesirable levels ofaggregation of the AAT. The amount of potassium acetate is preferably atleast 4%, more preferably at least 5%, and most preferably about 6% byweight.

[0076] After viral reduction, the AAT solution may optionally be dilutedand ultrafiltered, then re-concentrated and sterilized, e.g. byfiltration. The sterilized AAT-containing concentrate may then belyophilized to form a therapeutic product. A suitable composition forpreparing a lyophilized AAT powder is shown in Table 1. TABLE 1Composition of AAT solution for lyophilization Concentration ComponentFunction 1.0 g/vial AAT^(a) Active Ingredient 50 mg/mL^(b) SodiumPhosphate^(c) Buffer, Tonicity 20 mM Sodium Chloride USP Tonicity 40 mMMannitol USP Stabilizing Agent 3% Sodium Hydroxide To adjust pH asneeded Hydrochloric Acid ACS To adjust pH as needed Water for InjectionUSP^(d) Diluent/Vehicle 20 ml/vial

[0077] The final formulation will depend on the viral inactivationstep(s) selected and the intended mode of administration. Depending onwhether the AAT is to be administered by injection, as an aerosol, ortopically, the AAT may be stored as a lyophilized powder, a liquid, or asuspension. The composition shown in Table 1 is suitable for injection,and may be lyophilized and stored in glass vials for laterreconstitution with sterile water. The composition of a suitable drypowder formulation for inhalation is shown in Table 2. Such aformulation is suitable for inhalation administration as described inU.S. Pat. No. 5,780,014, either with a metered dose inhaler, or with apulmonary delivery device such as is disclosed in U.S. Pat. No.6,138,668. TABLE 2 Composition of AAT Formulation for AerosolAdministration Nominal Content Component Function (per unit dose) AATActive Ingredient  7.440 mg* Sodium Citrate Buffer 0.059 mg Citric AcidBuffer 0.001 mg

[0078] Assays for determining the quantity and quality of AAT are knownin the art and may be employed for evaluating the efficiency of themethod. An example of an immunoassay involving a monoclonal antibodyspecific for AAT, used for measuring or detecting AAT in biologicalfluids, is disclosed in U.S. Pat. No. 5,114,863. An example of the useof rate nephelometry is disclosed in L. Gaidulis et al., Clin. Chem.29:1838 (1983). AAT functional activity may be assayed by measuring itselastase inhibitory capacity using a chromogenic substrate for elastase,as described in U.S. Pat. No. 4,697,003. AAT may also be assayed bymeasuring its trypsin inhibitory capacity in a similar manner. In apreferred embodiment, AAT is assayed by endpoint nephelometry, asdescribed elsewhere in this specification.

[0079] The quantity of proteins may be determined by methods known inthe art, for example the Bradford assay, or by absorbance at 280 nnusing as an extinction coefficient E_(1 cm,280 nm) ^(1%)=5.3 (R.Pannell, D. Johnson, and J. Travis, Biochemistry 13:5439-5445 (1974)).SDS-PAGE with staining and densitometry may be used to assess purity ofthe sample and detect the presence of contaminating proteins. A reducingagent such as dithiothreitol is preferably used with SDS-PAGE to cleaveany disulfide-linked polymers, thereby facilitating the comparison oftotal AAT to total non-AAT protein. Size-exclusion HPLC may also be usedto assess purity of the sample and detect the presence of bothcontaminating proteins and aggregate or polymeric forms of AAT. Analysisof four lots prepared by the method of the invention showed AAT proteinpurity by SDS-PAGE (reduced) of at least 98%, an AAT monomer content ofat least 95%, and specific activity averaging 1.06 mg functional AAT/mgprotein (Table 3). TABLE 3 Purity of AAT % AAT Purity Specific Activityby SDS-PAGE % Monomeric AAT (mg functional Lot (reduced) by HPLC AAT/mg)A 98 95 1.10 B 99 95 1.09 C 98 95 1.05 D 98 96 1.04

[0080] Preferred conditions for the methods of the invention are asfollows:

[0081] 1. Preparation of Cohn Fraction IV₁₋₄.

[0082] Human plasma is cooled to −2 to 2° C. and adjusted to a pH of 6.9to 7.5. Cold ethanol is added to a concentration of 6 to 10%, and thetemperature is lowered to −4 to 0° C. The precipitate that forms(“Fraction I”) is removed by centrifugation or filtration.

[0083] The filtrate or supernatant from the above procedure is adjustedto pH 6.7 to 7.1, and cold ethanol is added to a concentration of 18 to22%. The temperature is lowered to −7 to −3° C., and the mixture isagain subjected to centrifugation or filtration. The precipitate thatforms (“Fraction II+III”) is set aside for other purposes.

[0084] The filtrate or supernatant from the above procedure is adjustedto pH 4.9 to 5.3 and the ethanol concentration is adjusted to 16 to 20%.The temperature is adjusted to −7 to 3° C. After the suspension settles,it is adjusted to pH 5.7 to 6.1 and the ethanol concentration isadjusted to 40 to 44%. The precipitate that forms (“Fraction IV₁₋₄”) isremoved by centrifugation or filtration, and stored until needed in theform of a paste. Fraction IC₁₋₄ contains AAT as well as contaminatingproteins and lipids.

[0085] 2. Purification with DTT and Silica.

[0086] The Fraction IV₁₋₄ paste is suspended in a suspension buffer(e.g., 100 mM Tris, 20 mM NaCl, pH between about 7.5 and about 9.5,preferably between about 8 and about 9) and stirred for a minimum of onehour at low temperature. The amount of buffer used ranges from 6 to 10kg of buffer per kg of the plasma-containing fraction.

[0087] The Tris buffer suspension is then treated with dithiothreitol(DTT) and fumed silica. DTT is added to the Tris buffer suspension at aconcentration in the range of about 10-50 mM. The solution is stirredfor at least 30 minutes, preferably 2-4 hours, at low temperature, andpreferably at a pH of about 8-9. Fumed silica is added at aconcentration of approximately 100-300 g fumed silica per kg Fraction IVprecipitate. The suspension is stirred for at least 30 minutes,preferably 1-4 hours, at low temperature, at a pH of about 8-9. A filteraid such as Celite™ is added at the rate of five parts filter aid onepart silica, by weight, and the mixture is stirred for approximately 15minutes. The soluble AAT product is separated from the precipitatedfumed silica and contaminating proteins using a filter press, yieldingthe AAT final filtrate. Preferably, the suspension is recirculatedthrough the filter press until the desired level of clarity is obtained.The AAT final filtrate is then treated further as follows.

[0088] 3. Ion Exchange Chromatography.

[0089] The AAT final filtrate is applied directly onto a chromatographycolumn containing an anion exchange resin equilibrated with an IECequilibration buffer. Contaminants are removed from the column bywashing the column with an IEC wash buffer, and AAT is subsequentlyeluted using an IEC elution buffer.

[0090] 4. Hydrophobic Interaction Chromatography (HIC).

[0091] The eluate from the IEC column is prepared for HIC by addingammonium sulfate to a final concentration of about 1 M. The solution isthen filtered and applied to a hydrophobic interaction chromatographycolumn which is equilibrated in a HIC wash buffer. Initial elution witha wash buffer provides an AAT-containing effluent, and elution withadditional wash buffer removes any AAT retained on the column. Thecombined effluent and washes are concentrated by ultrafiltration, anddiafiltered into a phosphate buffer. The final AAT concentration ispreferably no greater than 7% protein.

[0092] 5. Pasteurization

[0093] The AAT concentrate is stabilized for pasteurization by theaddition of sucrose and potassium acetate, and pasteurized at about 60°C. for 10-11 hours. The pasteurized solution is held at 2-8° C. pendingfurther processing.

[0094] 6. Nanofiltration

[0095] The pasteurized AAT solution is diluted with a final formulationbuffer. The diluted, pasteurized AAT solution is then filtered throughtwo new YM-100 (Amicon) spiral-wound ultrafiltration cartridges. Thisnanofiltration step serves as a second primary viral reduction step.Viruses are retained by the membrane, which has a nominal 100,000 Daltonmolecular weight cut-off, while AAT, which has an approximate molecularweight of 50 kD, passes through. The AAT is collected in the permeate ofthe second filter and in filter post-washes. The final filtrate iscollected in a bulk receiver and held at 2-8° C.

[0096] 7. Sterile filtration and lyophilization

[0097] The AAT-containing final filtrate is concentrated and diafilteredinto final formulation buffer at a temperature of no more than 15° C. toform a final bulk solution. This solution is clarified and sterilized bypassage through a series of sterile, bacterial-retentive filters. Thesterile bulk solution is filled into sterilized glass final containers.The filled containers are freeze-dried and then sealed under vacuum.

[0098] The product is ≧96% pure AAT as determined by both SDS-PAGE andimmunological assays such as ELISA or nephelometry, and is ≧93% monomerby size exclusion HPLC. The recovery based on the functionally activeAAT content of the Cohn fraction IV paste is 70%.

EXAMPLES

[0099] Fraction IV₁₋₄ Precipitate (667 kg) was isolated via the Cohnplasma fractionation process from 9026 liters of human plasma. Thematerial was divided into nine batches of approximately 75 kg each. Eachbatch was suspended in Tris Buffer, using 6 to 10 parts buffer (w/w)relative to the presscake. The suspensions were stirred for at least 15minutes, the temperature was adjusted to 2°-8° C., and the pH of eachsuspension was adjusted to 8.80-8.95 with 1 N sodium hydroxide or 1 Nhydrochloric acid as necessary. The suspensions were stirred for 15 to105 minutes (average 45 min), and monitored for protein (Bradford assay)and potency. Specific activity of each batch ranged from 0.027 to 0.045,and averaged 0.037 mg functional AAT per mg protein. Approximately 12%of the total protein was albumin, and approximately 22% was transferrin.

[0100] Dithiothreitol (DTT) was added to a final concentration of 0.01to 0.05 M DTT (average 0.03 M) based upon the amount of Tris Buffer ineach batch. After a pre-mix period of at least 15 minutes, thetemperature was adjusted to 2°-8° C. and the pH re-adjusted to8.80-8.95, and the solutions were stirred for 2 to 8 hours (average 3hours). If necessary, the pH was again adjusted to 8.80-8.95.

[0101] Aerosil™ 380 (Degussa AG, Frankfurt-Main) was added at the rateof 13.4 to 18.6 g per liter plasma input (average 16.7 g). Thesuspensions were stirred for 1 to 4 hours (average 1 hour) at 2-8° C.

[0102] Celite™ 545 was added to each suspension at the rate of 5 partsCelite to 1 part Aerosil, and the suspensions were stirred at 2-8° C.Each suspension was then recirculated through a plate and frame filterpress, holding 25×25 inch Cuno™ A2605-10CP filter pads (cellulose padswith inorganic filter aids; nominal cutoff 1 micron). When the turbiditywas ≦10 NTU by nephelometry (minimum of 15 min.), re-circulation wasdiscontinued and the filtrate was collected. The filter press waspost-washed with TRIS extraction buffer at 2-8° C. The postwashes werecombined with the initial filtrate solutions, and total protein insolution was determined by the Bradford protein assay. The filtrateswere held at 2-8° C. for no longer than 19 hours. Based on AAT activity,the filtrates contained a total of 1557 g of ATT, corresponding to a 59%yield of the activity present in the original suspension of Fraction IVpaste, and a purification factor of 1.5. (In view of the activitypresent after subsequent processing, these values appear to be low,possibly due to the presence of unidentified factors interfering withthe AAT assay.) Specific activity for each of the nine batches rangedfrom 0.042 to 0.064, and averaged 0.056 mg functional AAT per mgprotein. Albumin and transferrin were below detection limits (totalprotein contained less than 0.5% albumin and less than 2.5%transferrin.)

[0103] A 92-liter, 30 cm high ion exchange chromatography (IEC) columnloaded with TMAE Fractogel™ (EM Industries, Hawthorne, N.Y.) wasequilibrated with IEC equilibration buffer (50 mM Tris, pH 8.3-9.3,20-25° C.). Following equilibration, conductivity of the effluent wasverified to be ≦1.25 mS/cm. Each filtrate from the previous step waswarmed to 20-25° C. and filtered through a Cuno Zeta Plus™ 90SPcartridge (45115-12-90SP, nominal MW cutoff of 0.1 micron) beforeloading onto the column with control of flow rate (≦3.0 cm/minute) andcolumn pressure (≦20 psi). Total protein loaded onto the IEC column waslimited to no more than 70% of the resin capacity. The column was thenwashed with five column volumes of IEC wash buffer (50 mM Tris, 25-70 mMNaCl gradient, pH 7.1-7.7) at 20-25° C., with control of flow rate (≦3.0cm/minute) and column pressure (≦20 psi). The effluent was monitored byBradford protein determination, assay of AAT activity, and UV absorbanceat 280 nm.

[0104] AAT was eluted with approximately three column volumes of IECelution buffer (50 mM Tris, 75-120 mM NaCl gradient, pH 7.1-7.7) at20-25° C., with control of flow rate (≦3.0 cm/minute) and columnpressure (≦20 psi). The effluent was monitored by Bradford proteindetermination, assay of AAT activity, and UV absorbance at 280 nm. Theentire peak that eluted after application of the elution buffer wascollected for further processing.

[0105] The above procedure was repeated nine times in order to processall nine batches of filtrate. Ammonium sulfate was added to the IECeluates to a final concentration of 0.9 to 1.1 M. The resultingsolutions were either used immediately, or stored at 15-25° C. for nomore than seven days. Based on AAT activity, the IEC eluates contained atotal of 2241 g of ATT, corresponding to an 84% yield of the activitypresent in the original suspension of Fraction IV paste, and apurification factor of 16.2. Specific activity for each of the ninebatches ranged from 0.416 to 0.975, and averaged 0.592 mg functional AATper mg protein.

[0106] A Cuno™ filter (Zeta Plus™ 90SP cartridge (45115-12-90SP, nominalMW cutoff of 0.1 micron) was prepared with a hot WFI flush followed by acold WFI rinse (WFI=Water for Injection). Water was gently blown out ofthe filter with compressed air. Three IEC eluates, containing ammoniumsulfate, were pooled and filtered through the prepared Cuno filter andsubsequently combined to provide the “filtered IEC solution”. The filterwas post-washed with approximately 20 liters HIC wash buffer (50 mMTris, 1 M ammonium sulfate, pH 7.1-7.7). The post-wash and the filtratewere combined and weighed. The process was repeated three times toprocess the nine batches of IEC eluate.

[0107] A hydrophobic interaction column (HIC) was packed with PhenylSepharose™ Fast Flow HS resin (Pharmacia, Piscataway, N.J.) to a volumeof 49 liters (32 cm bed height), and equilibrated with HIC wash buffer(50 mM Tris, 1 M ammonium sulfate, pH 7.1-7.7). This and all columnloading and subsequent elutions were carried out with control of flowrate (≦4 cm/minute), column pressure (≦20 psi), and solutiontemperatures (20-25° C.).

[0108] Each of the three batches of filtered IEC solution was loadedonto an HIC column. Total protein load onto the column was limited to≦39 g protein per liter of resin. Optical density (OD₂₈₀) of theeffluent was monitored, and collection was initiated when the OD₂₈₀ rose0.04 units higher than the baseline value. The column was washed withHIC wash buffer to elute additional AAT from the column, while non-AATcontaminants remained bound to the column. Approximately ten columnvolumes of HIC wash buffer was applied to the column, and effluent wascollected until the A₂₈₀ dropped to <0.05 units above baseline. The AATeffluent and column wash were combined and weighed. Samples were takenfor Bradford protein determination, OD Protein determination, potency,and LAL (Limulus amebocyte lysate) testing. The HIC effluents were heldat 15-25° C. for no more than 72 hours. Based on AAT activity, the threebatches of HIC effluent contained a total of 2090 g of ATT,corresponding to a 79% yield of the activity present in the originalsuspension of Fraction IV paste, and a purification factor of 25.6.Specific activity for each of the three batches ranged from 0.908 to0.986, and averaged 0.937 mg functional AAT per mg protein.

[0109] A tangential flow ultrafiltration (UF) unit containing apolyether sulfone membrane (surface area: 50 ft²) with a molecularweight cut off range of 5,000-30,000 was integrity tested to ensure abubble point of less than 1250 ml/minute. Diafiltration buffer (40 mMsodium phosphate, pH 7.2-7.6; 10 kg minimum) was recirculated throughthe unit for a minimum of five minutes. The recirculated buffer solutionwas sampled to verify proper pH (7.2-7.6) and LAL (<0.25 EU/ml). Arepeat of the prewash steps was performed if pH and LAL requirementswere not met. The UF unit was held for no more than 12 hours at 2-8° C.prior to HIC Effluent application.

[0110] The HIC effluent from the previous process step was mixed, andthe temperature was adjusted to 15-25° C., prior to application to theultrafiltration unit. Inlet pressure was maintained at ≦40 psi, andoutlet pressure and sample weight were monitored during theconcentration process. Concentration was performed until the weight ofthe concentrate was approximately 10 kg.

[0111] Following concentration, the HIC effluent concentrate wasdiafiltered, exchanging the Tris-buffered ammonium sulfate solution witha sodium phosphate buffer. Diafiltration buffer (40 mM sodium phosphate,pH 7.2-7.6) was applied at a volume ten times the weight of the HICeffluent concentrate. Inlet pressure was maintained at <40 psi, andoutlet pressure was monitored. After all of the diafiltration buffer hadbeen added, the sodium concentration of the permeate was determined.Diafiltration was considered complete if the sodium concentration of thepermeate was within 10% of that of the diafiltration buffer. Additionaldiafiltration buffer (5× the weight of the concentrate) was added, anddiafiltration extended, if necessary, until the sodium concentration ofthe permeate was within ±0% of that of the diafiltration buffer.

[0112] Following diafiltration, the ultrafiltration was continued untilthe concentrate had a mass of approximately 6 kg. Product was thengently blown out of the UF system (≦25 psi). The ultrafiltration unitwas postwashed twice with 1.5 kg diafiltration buffer. The UF postwasheswere added to the diafiltered concentrate. The total weight ofconcentrate was determined and the protein concentration determined (ODat 280 nm).

[0113] Based on the OD protein observed, the AAT protein concentrationwas determined, and adjusted if necessary to the range 2.9-6.8%.Analysis for LAL, SDS-PAGE, Bradford protein, potency, and bioburdenwere performed. SDS-PAGE showed ≧98% AAT. Based on AAT activity, theconcentrates contained a total of 2096 g of AAT, a 79% yield of theactivity present in the Cohn paste suspension, and a purification factorof 26.6. Specific activity for each of the three batches ranged from0.886 to 1.04, and averaged 0.974 mg functional AAT per mg protein.

[0114] The AAT concentrate (2.9-6.8% protein) was adjusted to 20-25° C.,and sucrose (1.75 kg per kg AAT concentrate) and potassium acetate(0.175 kg per kg AAT concentrate) were added. The final concentration ofsucrose was 59.8%+6% (w/w), and the final concentration of potassiumacetate was 5.98%±0.6% (w/w). After mixing, the stabilized concentratewas transferred into one-liter sealed serum bottles. The bottles werestored at 2-8° C. for no more than 10 weeks (and at 15-25° C. for nomore than 48 hours) before being heat-treated (pasteurized).Pasteurization at 60±1° C. was performed for 10-11 hours. Thepasteurized AAT solution was held at 2-8° C. for no more than 10 weeks,and at 15-25° C. for no more than 72 hours, prior to further processing.

[0115] Pasteurized AAT solution was pooled under HEPA-filtered air intotwo batches, and diluted with diafiltration buffer (20 mM sodiumphosphate, 45 mM NaCl, 3% mannitol, pH 6.6-7.4) at a ratio of 5:1buffer:AAT solution (w/w). The diluted solutions were sampled for LAL,protein, and potency. Based on AAT activity, the pasteurized and dilutedsolutions contained a total of 1941 g of AAT, a 73% yield of theactivity present in the Cohn paste suspension, and a purification factorof 26.6. Specific activities for the two pasteurized batches were 0.954and 0.993, an average of 0.973 mg functional AAT per mg protein. Thepercent monomer of the AAT solutions was measured by size-exclusion HPLCbefore and after pasteurization. The monomer fractions of the AATconcentrates (pre-pasteurization) were 97.1% to 98.5%, averaging 97.7%.The monomer fractions of the two pasteurized and diluted solutions were95.9% and 97.5%, an average of 96.7%. Only 1.0% of the monomeric form ofAAT was polymerized or aggregated during the pasteurization step.

[0116] Two YM100 filter cartridges (Millipore, Bedford, Mass.) wereinstalled in series into a YM100 UF system, with the first cartridgeoperated in a tangential flow mode and the second cartridge dead-ended.The UF system was recirculated with a minimum of 5 kg diafiltrationbuffer. Following recirculation, the diafiltration buffer was tested toverify pH (6.8-7.2) and LAL (<0.25 EU/ml). The diafiltration buffer, andall subsequent processing until lyophilization, was at 2-8° C.

[0117] Each of the pooled AAT solutions was passed through the YM100cartridges at 2-8° C. at an inlet pressure of ≦45 psi. The load did notexceed 1339 grams protein, and the weight of the YM100 filtrate pluspostwashes did not exceed 337 kg. The YM100 filtrates were thenultrafiltered and diafiltered, at an inlet pressure of ≦50 psi, againstdiafiltration buffer (1.60-1.90 mg/ml sodium, 10 times the YM100concentrate weight), using an ultrafilter containing a 10,000 M.W.membrane (≧25 ft² surface area) that was dedicated to thepost-pasteurization process.

[0118] The diafiltered solutions were sampled inline and tested forsodium. If the sodium level of the permeate was within ±10% of thediafiltration buffer sodium concentration, diafiltration was consideredcomplete. If the sodium level was not within ±10% of the diafiltrationbuffer sodium concentration, diafiltration was repeated with additionaldiafiltration buffer (5 times the YM100 filtrate weight).

[0119] A final concentration was performed until approximately 6 kg ofsolution was obtained. Two postwashes were performed using 1.5 kgdiafiltration buffer each time. Postwashes were combined with theconcentrate for determination of total volume of diafiltered YM100filtrate. Diafiltered YM100 filtrates were held for no more than 12 daysat 2-8° C. before further processing. Based on AAT activity, thediafiltrate contained a total of 1960 g of AAT, a 74% yield of theactivity present in the Cohn paste suspension, with a purificationfactor of 27.5. Specific activities for the two batches were 0.984 and1.03, an average of 1.01 mg functional AAT per mg protein.

[0120] After addition of diafiltration buffer to obtain a finalformulation target of 50 mg functional AAT/ml, the YM100 filtratesolution pH was adjusted as necessary to pH 6.8-7.2. Clarification wascarried out with a 0.2 micron Pall SLK-7002-NRP Filter (Pall Corp., EastHills, N.Y.). Once clarified, the non-sterile bulk AAT solutions werecombined, weighed and sampled for LAL, protein, potency, and bioburden(<100 CFU/ml). The non-sterile bulk AAT was held for no longer than 73.5hours at 2-8° C. pending sterile filtration. Based on AAT activity, thenon-sterile bulk AAT solution contained a total of 1822 g of AAT, a 69%yield of the activity present in the Cohn paste suspension, with apurification factor of 26.8. The specific activity was 0.981 mgfunctional AAT per mg protein.

[0121] In preparation for sterile filtration, a sterile bulk assemblyconsisting of a 60 L bulk receiver, a Pall 0.2 micron KA1NFP2sterilizing filter and two (2) Millipore 0.2 Micron Aervent™ 50 ventfilters was prepared. The assembly was autoclaved and used within 7 daysof autoclaving. The non-sterile bulk solution was sterile-filtered withcontrol of temperature (2-8° C.), pressure (≦20 psi), filtration time(≦120 minutes), and load including postwash (≦0.26 kg non-sterile bulkper cm² filter area). The sterile filtrate ultimately obtained from 667kg of Cohn fraction IV paste contained 1.78 kg of functional AAT,corresponding to an overall yield of 67% based on the activity of theinitial Cohn fraction IV₁₋₄ suspension, and a purification factor of29.8. The specific activity was 1.09 mg functional AAT per mg protein.The product was >99% AAT by SDS-PAGE, and >95% monomer by size-exclusionHPLC.

[0122] AAT sterile bulk was aseptically filled into 50 ml Type I glassvials using a fill volume targeted to achieve approximately 1000 mgfunctional AAT activity per vial (i.e. 20.8 g±0.2 g solution per vial),and the vial contents were frozen and lyophilized. TABLE 4 Fr. IV_(1,4)Post- IEC HIC DF HIC Diluted, YM100 Non-Sterile Final Extract AerosilFiltrate Eluate Effluent Conc. Pasteur. Filtrate Bulk Container No. ofBatches 9  19  9 9 3 3 2 2 1 1 Yield (g AAT; 2658 1833* 1557* 2241 20902096 1941 1960 1822 1780 total for all batches) Overall Yield 100%  69% 59% 84% 79% 79% 73% 74% 69% 67% from Extract Purification 1.0    1.4   1.5 16.2 25.6 26.6 26.6 27.5 26.8 29.8 Factor Specific 0.037^(†)     0.053^(†)      0.056^(†) 0.592^(†) 0.937^(‡) .0974^(‡) 0.973^(‡)1.01^(‡) 0.981^(‡) 1.09^(‡) Activity** (mg/mg)

[0123] Functional AAT yields, and characteristics of the AAT fractionsobtained, at each of the above steps are set out in Table 4.Modifications of the above-described modes for carrying out theinvention will be obvious to those of skill in the fields of proteinpurification, analytical chemistry, medicine, and related fields, andsuch substitutions and modifications are contemplated to be within thescope of the invention. The detailed embodiments described above areprovided by way of example only, and are not intended to limit the scopeof the following claims.

We claim:
 1. A method for partially purifying AAT from an AAT-containingprotein mixture, consisting essentially of: (a) suspending theAAT-containing protein mixture in a buffer under conditions that permitthe AAT to be dissolved; (b) contacting the resulting suspension with adisulfide-reducing agent to produce a reduced suspension; (c) contactingthe reduced suspension with an insoluble protein-adsorbing material; and(d) removing insoluble materials from the suspension.
 2. A method forpurifying AAT from a crude AAT-containing protein precipitate,comprising the steps of: (a) suspending a crude AAT-containing proteinprecipitate in a buffer under conditions that permit the AAT to bedissolved; (b) contacting the resulting suspension with adisulfide-reducing agent to produce a reduced suspension; (c) withoutaddition of a substantial amount of additional salts, contacting thereduced suspension with an insoluble protein-adsorbing material; and (d)removing insoluble materials from the suspension.
 3. The method of claim1, wherein the crude AAT-containing protein precipitate is derived fromserum.
 4. The method of claim 2, wherein the crude AAT-containingprotein precipitate is derived from serum.
 5. The method of claim 3,wherein the crude AAT-containing protein precipitate is Cohn fractionIV₁₄ precipitate.
 6. The method of claim 4, wherein the crudeAAT-containing protein precipitate is Cohn fraction IV₁₋₄ precipitate.7. The method of any of claims 1-6, wherein the disulfide-reducing agentis a dithiol.
 8. The method of claim 7, wherein the dithiol isdithiothreitol.
 9. The method of any of claims 1-6, wherein theprotein-adsorbing material is a silica adsorbent.
 10. The method of anyof claims 1-6 wherein the protein-adsorbing material is fumed silica.11. The method of claim 7, wherein the protein-adsorbing material isfumed silica.
 12. The method of claim 8, wherein the protein-adsorbingmaterial is fumed silica.
 13. The method of claim 2, further comprisingan anion exchange chromatography step.
 14. The method of claim 3,further comprising an anion exchange chromatography step.
 15. The methodof claim 13, further comprising a hydrophobic interaction chromatographystep.
 16. The method of claim 14, further comprising a hydrophobicinteraction chromatography step.
 17. The method of claim 15, furthercomprising a viral reduction step.
 18. The method of claim 16, furthercomprising a viral reduction step.
 19. The method of claim 17, whereinthe viral reduction step comprises pasteurization at about 60° C. 20.The method of claim 18, wherein the viral reduction step comprisespasteurization at about 60° C.
 21. The method of claim 20, wherein thepasteurization step is carried out on a solution of AAT containing atleast 40% w/w sucrose.
 22. The method of claim 21, wherein theconcentration of sucrose is at least 50%.
 23. The method of claim 22,wherein the concentration of sucrose is about 60%.
 24. The method ofclaim 19, wherein the viral reduction step further comprises filtrationeffective to remove viral particles.
 25. The method of claim 23, whereinthe viral reduction step further comprises filtration effective toremove viral particles.
 26. The method of any one of claims 15-25,further comprising a sterilization step.
 27. The method of claim 26,wherein the sterilization step comprises filtration effective to removebacteria.
 28. A method for purifying AAT from a crude AAT-containingprotein precipitate, comprising the steps of: (a) suspending anAAT-containing Cohn fraction IV₁₋₄ precipitate in a buffer underconditions that permit the AAT to be dissolved; (b) contacting theresulting suspension with dithiothreitol to produce a reducedsuspension; (c) without addition of a substantial amount of additionalsalts, contacting the reduced suspension with fumed silica; (d) removinginsoluble materials from the suspension to obtain a clarified proteinsolution; (e) anion exchange chromatography of the clarified proteinsolution; (f) hydrophobic interaction chromatography of the product ofthe anion exchange chromatography; (g) pasteurization of the product ofthe hydrophobic interaction chromatography at about 60° C.; (h)filtration of the pasteurized product effective to remove viralparticles; and (i) sterile filtration.
 29. An alpha-1-antitrypsincomposition, containing: (a) less than 6% contaminating proteins bySDS-PAGE; (b) less than 0.1% albumin; (c) less than 0.8% α₁-acidglycoprotein; (d) less than 0.1% α₂-macroglobulin; (e) less than 0.1%apolipoprotein A1; (f) less than 0.5% antithrombin III; (g) less than0.1% ceruloplasmin; (h) less than 0.5% haptoglobin; (i) less than 0.2%IgA; (j) less than 0.1% IgG; and (k) less than 0.1%. transferrin;wherein the specific activity of the alpha-1-antitrypsin is at least0.99 mg functional AAT per milligram, when using as an extinctioncoefficient E_(1 cm,280 nm) ^(1%)=5.3; wherein the apparent ratio ofactive to antigenic AAT is greater than 1.08, when measured by endpointnephelometry; wherein less than 8% of the composition is of a highermolecular weight than monomeric AAT; and wherein the composition isstable for at least 2 years when stored lyophilized at up to 25° C. 30.The composition of claim 29, wherein the number of enveloped viruses arereduced by at least 11 log₁₀ units and non-enveloped viruses are reducedby at least 6 log₁₀ units.
 31. The composition of claim 29 or claim 30,wherein the composition contains less than 2% contaminating proteins bySDS-PAGE, and wherein the apparent ratio of active to antigenic AAT isgreater than 1.16.
 32. The composition of claim 31, wherein thecomposition contains (a) less than 1% contaminating proteins bySDS-PAGE; (b) less than 0.2% α₁-acid glycoprotein; (c) less than 0.1%antithrombin III; (d) less than 0.1% haptoglobin; and (e) less than 0.1%IgA; and wherein less than 5% of the composition is of a highermolecular weight than monomeric AAT, and the apparent ratio of active toantigenic AAT is greater than 1.23.